Identification of the Toxic Pentapeptide Nodularin in A

A tica nal eu yt c ic a a m A r a c

h a P Pacheco et al., Pharm Anal Acta 2016, 7:5 Pharmaceutica Analytica Acta DOI: 10.4172/2153-2435.1000479 ISSN: 2153-2435

Research Article Open Access

Identification of the Toxic Pentapeptide Nodularin in a Cyanobacterial Bloom in a Shrimp Farm in South American Atlantic Coast Pacheco LA1,3, Kunrath N1, Costa CM1,4, Costa LDF1, Foes GK2, Wasielesky W2 and Yunes JS1* 1Laboratory of Cyanobacteria and Phycotoxins, Institute of Oceanography, Federal University of Rio Grande, RS, Brazil 2Aquaculture Marine Station (EMA), Institute of Oceanography, Federal University of Rio Grande, RS, Brazil 3Post Graduate Program in Physical, Chemical and Geological Oceanography , Institute of Oceanography, Federal University of Rio Grande, RS, Brazil 4Post Graduate Program in Aquaculture, Institute of Oceanography, Federal University of Rio Grande, RS, Brazil *Corresponding author: Yunes JS, Laboratório de Cianobactérias e Ficotoxinas, IOFURG, Universidade Federal do Rio Grande, 96.203-270 - Rio Grande, RS, Brazil, Tel: +55 53 32336737; E-mail: [email protected] Received date: Apr 28, 2016; Accepted date: May 23, 2016; Published date: May 25, 2016 Copyright: © 2016 Pacheco LA et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Abstract

Since 2010, blooms of the brackish cyanobacteria Nodularia spumigena are recurrent in the shrimp growth tanks of the Marine Aquaculture Station during summer in Southern Brazil. Cyanobacterial growth led to a decrease in the white shrimp Litopenaeus vannamei productivity. In the summer of 2014, a Nodularia bloom was collected from the tanks; filaments were separated by flotation and washed thoroughly twice in F/2 culture medium. Healthy filaments were lyophilized and the powder used for nodularin quantification by HPLC-DAD and immunoassays. Nodularin containing lyophilized powder was also tested for toxicity against the brine shrimp Artemia salina post-larvae and the white shrimp Litopenaeus vannamei 35 days old larvae. The lyophilized Nodularia powder contained 1.88 mg of the toxin nodularin g-1d.w. Its toxicity was confirmed in bioassays with Artemia salina and Litopenaeus vannamei giving -1 a LC50 of 1.22 and 2.50 µgL of nodularin, respectively. This paper firstly describes the occurrence and the toxicity of nodularin in South Atlantic coastal waters with consequences to shrimp farming.

Keywords Cyanobacteria; Litopenaeus vannamei; Nodularia Later, dog and cattle poison have also resulted from nodularin spumigena; Nodularin; Shrimp farm; Toxicity tests ingestion in the Baltic Sea [3,4], and more specifically in the gulf of Finland, considerable losses in the North Atlantic flounder Platichtlys Introduction flexus populations were documented following a Nodularia bloom collapse [2]. Nodularia cells produce the pentapeptide nodularin Early registration of scum, or colored waters, consistent with whose toxicity and lethal concentration (i.p.) in mammals are similar cyanobacterial blooms refer back to at least 1853. In a perceptive and to microcystins [5,6]. The main target organ of nodularin and prescient paper in Nature, the Adelaide assayer and chemist George potentially MC-LR is the liver [7], which expresses high levels of many Francis reported on stock deaths at Milang on the shores of Lake uptake transporters, including Oatps [8]. One of the important Alexandrina in South Australia. Francis attributed the deaths to the molecular toxicological mechanisms of these toxins is the inhibition of ingestion and toxicity of scums of the cyanobacterium Nodularia serine/threonine specific protein phosphatases PP1 and PP2A [9]. This spumigena [1]. in turn leads to hyperphosphorylation of proteins, ultimately resulting in deterioration of cellular integrity. A marine shrimp farm located on the south coast of Brazil (Cassino Beach, RS, Brazil) uses the "bioflocs" system of intensive cultivation, in order to achieve a high production of shrimp associated with high air flow with this microbial aggregate and without water exchange [10]. The microbial aggregation was stimulated by the extra addition of organic carbon sources; however, at outdoors shrimp tank systems this carbon input stimulates photoautotrophic organisms. Moreover, shrimp excrements and excess food portions, under sunlight exposure (open systems), led to a microbial growth [11]. Due to its geographical location the Aquiculture Marine Station (EMA) from the Federal University of Rio Grande (32º12’S, 52º10’W) runs the intensive shrimp (Litopenaeus vannamei) growth mainly at the end of spring and before autumn. In this period high illumination Figure 1: Open shrimp tanks next to the shoreline at the Cassino and drought turns environmental conditions ideal for the shrimp beach where EMA is located. Intense blue-green color of growth. On the other hand, these were also ideal conditions for cyanobacterial blooms in the water. cyanobacterial (Nodularia spumigena) growth leading to a decrease on shrimp growth and survival in some outdoors tanks.

Pharm Anal Acta, an open access journal Volume 7 • Issue 5 • 1000479 ISSN: 2153-2435 Citation: Pacheco LA, Kunrath N, Costa CM, Costa LDF, Foes GK, Wasielesky W and Yunes JS (2016) Identification of the Toxic Pentapeptide Nodularin in a Cyanobacterial Bloom in a Shrimp Farm in South American Atlantic Coast. Pharm Anal Acta 7: 479. doi: 10.4172/2153-2435.1000479

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The presence of Nodularia filaments in the outdoor tanks was first 5.41.240 software. The analytical column used for analysis was a Luna identified and reported by Costa et al.[12] in the period above C18 (2), (250 x 4.6 mm, 5 μ) (Phenomenex®). The detection limit, described with the strong fall in productivity described in Table 1. resulting from SIGMA-Aldrich® standard analyzed by HPLC-DAD for nodularin was 0.025 µg L-1. Elisa analysis for NOD was also done using -1 PRODUCTIVITY (kg ha ) the specific immunoassay test for nodularins in the range from: 0.25 to -1 YEAR TANKS TANKS with NOD 1 μg L (Beacon® (ME, USA)) following instructions of the supplier.

2010 13600 7100 Toxicity tests 2011 5630 2200 NOD containing lyophilized powder was used in the toxicity assays against larvae of the brine shrimp Artemia salina and the 35 days old Table 1: Productivity of the white shrimp Litopenaeus vannamei in the post-larvae of the white shrimp Litopenaeus vannamei. Both tanks with, and without, Nodularia (NOD) blooms. organisms were supplied by the Marine Aquaculture Station (EMA- FURG). Both tests were run without water renovation, but with a strict However the precise estimation and identification of the toxin control of the nodularin contents in all triplicates during 24 h and 92 h content and toxicity to shrimps is necessary and this is the objective of length experiments, respectively. Thus, the NOD containing the present work. The present work also described and summarized lyophilized powder was used in tests at seven concentrations ranging methodological procedures which can be followed to monitor the from 0 to 5.0 mg powder per mL-1 in a 500 mL glass bottle with occurrence of this toxic pentapeptide in aquaculture farms. aeration for white shrimp Litopeaneus vannamei. The NOD containing lyophilized powder was also used in seven concentration tests ranging -1 Materials and Methods from 0 to 5.0 mg. mL for the brine shrimp Artemia salina in a 1 mL well of a 96 wells plate kept inside a 28oC incubation chamber. Cyanobacterial sampling Results The bloom analysed in this work occurred in the tank number 3 during the south hemisphere summer from 2013 to 2014. Samples Nodularin identification were collected using buckets and taken to the laboratory as a total volume of 10 liters. Floating filaments of Nodularia were separated Lyophilized powder from bloom in the tanks was used for from the water, algae and other organisms by the use of a slow identification of the presence of nodularin in the samples. Analysis by centrifugation LKB centrifuge at 2.000 rpm and separating a 2 liters High Performance Liquid Chromatography attached to a UV-DAD sample each time. Floating healthy filaments were predominant and detector after methanol extraction, and concentration, reveals the were pipeted into a 50µm phytoplankton net and fully washed 3 times presence of a single peak. A single peak was detected at a retention with the F/2 culture media prepared to marine cyanobacteria salinity. time from 6.1 to 6.3 minutes which corresponds to an identical peak of The resulting scum achieved 100% purity of Nodularia cells. The scum the nodularin standards supplied by Sigma-Aldrich® (Figure 2) or was frozen and totally transferred to a lyophilizer (Micromodulyo- Alexis Biochemicals®. Edwards®).

Sample treatment of nodularin analysis A 500mg of Nodularia lyophilized powder was weighted in a precision balance (Marte, Brazil). A 7 mL of Milli-Q water plus 8mL of methanol were added to it and let to extract during 24 h on an orbital shaker (Aros 16O, Thermolyne-USA®) at room temperature. The whole extract was transferred to a rotaevaporator and let to concentrate during 1 hour. The resulting sample was homogenized using an ultrasonic device (Hielsher, Germany®) three times during 30 seconds, with 30 seconds intervals. This sample final volume was collected into a glass cylinder and set to 6 mL, with the initial mixture of Milli-Q water:methanol (7:8). This extract was used and applied to the NOD concentration analysis by HPLC-DAD and immunoassays for microcystins and nodularins. The preparation of the solutions to HPLC-DAD was performed using only the reagents with HPLC grade and ultrapure Milli-Q water in preparing solutions. NOD standards and their reference material certificates were obtained from SIGMA-Aldrich® and from Abraxis® Figure 2: Chromatograms of the NOD bloom sample (a) and the (USA). The two mobile phases used were 0.05 M trifluoroacetic acid NOD standard (2b). (Merck®) in acetonitrile (Merck®) and 0.05 M acid trifluoroacetic in ultrapure Milli-Q water. The chromatograph HPLC-DAD (Shimadzu, Japan) consists of a controller CBM- 20A, a detector SPD-M20 Elisa analysis for nodularin (NOD): the Nodularia bloom was also analyzed for the presence of nodularin using the specific test for (200-400 nm), a deuterium lamp (D2), two binary pumps LC-20AD -1 and CTO-20A column oven at 40°C controlled through Labsolution nodularins in the range from: 0.25 to 1 μg L supplied by Beacon®

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(ME, USA). A value of 0.4 mg per liter of the dried lyophilized powder under risk of severe fish and crustacean intoxication as a result of a corresponds exactly to the highest value of the kit calibration interval. lack of a proper surveillance method, now available herein. Giving a positive response to the presence of nodularin in the sample This communication firstly describes the occurrence of toxic NOD and a possible nodularin concentration of 2.5 μg L-1 NOD mg dry in Brazilian waters with consequences to the shrimp farming in South lyophilized bloom powder. American Atlantic Coast. Toxicity tests References The toxicity of the Nodularia lyophilized powder was confirmed in Francis G (1878) Poisonous Australian Lake. Nature 18: 11-12. acute tests with the brine shrimp Artemia salina larvae. In three tests 1. performed using a minimum of a hundred organisms the lethal 2. Drevs TA, Jaanus E, Vahtmae (2007) Effects of the cyanobacterial blooms on the abundance of the flounder Platichthys flesus (L.) in the Gulf of concentrations which kills 50% of the organisms varied from 0.65 to Finland. Proc Estonian Acad Sci Biol Ecol 56: 196-208. 2.61 mg of the lyophilized Nodularia powder per mL of seawater. 3. Edler LMG, Ferno RL, Nilson PO (1985) Mortality of dogs associated Equally the same powder was tested against 35 days old post-larvae of with a bloom of the cyanobacterium Nodularia spumigena in the Baltic the white shrimp Litopenaeus vannamei giving a CL50 of 1.33 Sea. Ophelia 24: 103-109. -1 mg.mL . Taking into account that the nodularin concentration Kruger TS, Mundt DB, Luckas B (2009) Dog poisonings caused by -1 4. detected by HPLC in the powder was 1.885 mg g d.w. The nodularin Nodularia spumigena. Toxicol Lett 189: 207. amounts that killed 50% of Artemia and Litopenaeus larvae were 1.22 5. Carmichael WW (1989) characterization, pharmacology and and 2.50 µg L-1, respectively. therapeutics, In Natural toxins, Pergamon Press, Oxford, pp: 3-16. 6. Yoshizawa S, Matsushima R, Watanabe, MF, Harada, KI, Ichihara A Discussion (1990) Inhibition of protein phosphatases by microcystin and nodularin associated with hepatotoxicity. J Cancer Res Clin Oncol 116: 609-614. The present paper reports the development of Nodularia bloom 7. Svircev Z, Baltić V, Gantar M, Juković M, Stojanović D (2010) Molecular caused probably by the excess fertilized in the tanks of a shrimp farm. aspects of microcystin-induced hepatotoxicity and hepatocarcinogenesis. The bloom was strongly toxic to the white shrimp growth in the tanks J Environ Sci 28: 39-59. as well as in bench tests using a 35 day old post-larvae of the same 8. Roth M, Obaidat A, Hagenbuch B (2012) OATPs, OATs and OCTs: the shrimp. The reference marine toxicity test used, Artemia salina, also organic anion and cation transporters of the SLCO and SLC22A gene confirmed the Nodularia bloom toxicity. Using certified standards superfamilies. Br J Pharmacol 165: 1260-1287. supplied from two laboratories, a single peak of the pentapeptide 9. MacKintosh C, Beattie KA, Klumpp S, Cohen P, Codd GA (1990) Nodularin (NOD) was identified in the sample. Cyanobacterial microcystin-LR is a potent and specific inhibitor of protein phosphatases 1 and 2A from both mammals and higher plants. NOD is toxic pentapetide with a similar spectrum of action as the FEBS Lett 264: 187-192. hepatoxin microcystin to mammals [6]. The potencies of nodularins 10. Wasielesky W, Emerenciano M, Ballester S, Cavalli RRO, Abreu PC -1 (2006) Cultivos em Meios com Flocos microbianos. Panorama da and microcystins- LR are the same, having LD50 of 60 µg kg (ip, -1 Aquicultura 16: 14-23. mice) and a CL50-18h of 4.79 µg mL in Artemia salina [6,13]. Equally 11. Hargreaves JA (2013) Biofloc Production Systems for Aquaculture. SRAC an CL50-24h for Microcystis aeruginosa lyophilized powder to the local pink-shrimp Farfantepenaeus paulensis was 0.91 mg mL-1 [14]. The Publication 4503: 1-12. reports on their inhibitory activity against protein phosphatases 1 and 12. Costa C, Wasielesky W, Odebrecht C (2013) Impacto da Presença da Cianobacteria Nodularia spumigena em cultivo de camarões marinhos 2A render these compounds as important toxins to be aware [6]. em sistema de bioflocos. Reunião Latina Americana Sobre Algas Nocivas, However, as a typical brackish water growing organism, the pp: 7-9. cyanobacteria Nodularia impacted shallow areas of coastal regions in 13. Rinehart KL, Harada KI, Namikoshi M, Chen C, Harvis CA (1988) the Gulf of Finland [15] and in South Australian marine farms [16]. Nodularin, microcystin, and the configuration of Adda. J Am Chem Soc NOD has caused the death of several marine organisms, including 110: 8557-8558. fish of the genus Gaterosteus aculeatus and its concentration in the 14. Yunes JS (2009) Florações de Microcystis na Lagoa dos Patos e o seu estuário: 20 anos de Estudos. Oecol Bras 13: 313-318. animal tissues reached 170 µg NOD kg d.w. Also, it was suggested as Kankaanpää HT, Sipiä VO, Kuparinen JS, Ott JL,Carmichael WW (2001) the cause of the death of sea mullets reported in Australia. The NOD 15. Nodularin analyses and toxicity of a Nodularia spumigena (Nostocales, animal tissue concentration was 43.6 mg/kg [16]. Therefore, a proper Cyanobacteria) water-bloom in the western Gulf of Finland, Baltic Sea, in surveillance in the tissue of shrimp farm animals reported in this paper August 1999. Phycologia 40: 268-274. must be considered. 16. Stewart I, Eaglesham GK, McGregor GB, Chong R, Seawright AA (2012) While several environmental waters have been cited to host NOD First Report of a Toxic Nodularia spumigena (Nostocales/Cyanobacteria) Bloom in Sub-Tropical Australia. II. Bioaccumulation of Nodularin in producing cyanobacterial blooms with consequences to animal Isolated Populations of Mullet (Mugilidae). Int J Environ Res Public drinking, plant irrigation, bathing and leisure, aquaculture farmers are Health 9: 2412-2443.

Pharm Anal Acta, an open access journal Volume 7 • Issue 5 • 1000479 ISSN: 2153-2435